LOCUS       sequence-447746-        3599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for rhamnose inducible expression and purification of 
            protein A-Hia5-6XHis (pA-Hia5) for antibody directed DNA (adenine) 
            methylation (as in DiMeLo-seq).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3599)
  AUTHORS   Altemose N, Maslan A, Smith OK, Sundararajan K, Brown RR, Mishra R, 
            Detweiler AM, Neff N, Miga KH, Straight AF, Streets A
  TITLE     DiMeLo-seq: a long-read, single-molecule method for mapping 
            protein-DNA interactions genome wide.
  JOURNAL   Nat Methods. 2022 Jun;19(6):711-723. doi: 
            10.1038/s41592-022-01475-6. Epub 2022 Apr 8.
  PUBMED    35396487
REFERENCE   2  (bases 1 to 3599)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-022-01475-6"; journalName: "Nat Methods"; date: 
            "2022-06"; volume: "19"; issue: "6"; pages: "711-723"
COMMENT     Sequence Label: prhaBAD-pA-Hia5
FEATURES             Location/Qualifiers
     source          1..3599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..91)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(100..130)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        195..313
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     RBS             332..340
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             352..777
                     /codon_start=1
                     /product="immunoglobulin binding domains of Staphylocuccus 
                     aureus protein A"
                     /label=protein A
                     /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
                     PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
                     NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
     CDS             1687..1704
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1734..1752)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1748..1795
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1893..1922
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      2019..2607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2508..2527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2739..2766)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2790..3599)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3513..3532
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
       61 gtgaaagttg gaacctctta cgtgccgatc aagaagacgg tcaaaagcct ccggtcggag
      121 gcttttgact ttctgctatg gaggtcaggt atgatttaaa tggtcagtat tgagcgatat
      181 ctagagaatt cgtccaccac aattcagcaa attgtgaaca tcatcacgtt catctttccc
      241 tggttgccaa tggcccattt tcctgtcagt aacgagaagg tcgcgaattc aggcgctttt
      301 tagactggtc gtagagacca tgaaattctt ttaaggaggt aaaaaatgcc atcactgaaa
      361 gatgacccga gccagtctgc gaatctgttg agcgaagcga agaaactgaa cgaatctcaa
      421 gctccgaagg ccgacaacaa gtttaacaaa gagcagcaga acgcgtttta tgaaattctg
      481 catctgccga atttgaacga agaacagcgc aatggtttca ttcaatcctt gaaagatgat
      541 ccgagccaaa gcgcgaatct gctggccgag gcgaaaaagt tgaacgacgc acaggcaccg
      601 aaagccgata acaagttcaa caaagaacag caaaatgcgt tctatgaaat cctgcacttg
      661 ccgaatctga ccgaagaaca aagaaatggt tttatccaga gcctgaaaga cgaccctagc
      721 gtcagcaaag aaatcctggc agaggccaaa aagctgaatg acgcgcaagc gccgaaaggt
      781 gggggtggtt ctggcggtgg cggttctggt ggcggtggtt ccggtggtgg cggtagcatg
      841 gcaaatcaga atactttcaa acaagcaccg ctgccgttta tcggccagaa acgcatgttc
      901 ctgaaacagt tcgagcaaat tctgaacgaa aacatcagcg ataatggcga gggttggacg
      961 atcctggaca cgtttggcgg cagcggtttg ctgagccaca ccgcgaagcg cctgaagccg
     1021 aaggctcgtg tcatctataa cgatttcgat ggctacgccg aacgtctggc gcacatcgac
     1081 gacattaatc agctgcgtgc agagctttac agcgttgttg gcaatgcgac gagcaagaat
     1141 aaacgtatga ccaaagactg caaagccgag tgcattcgta tcattcagaa tttcaaaggc
     1201 tacaaagatc tgaactgtct ggcgtcctgg ctgctgttta gcggccaaca agtggcaact
     1261 ctggacgatc tgtttcagca caatttctgg cactgcattc gtcagagcga ctacccgaaa
     1321 gctgacggtt acctggacgg cgttgagatc gtcaaagaat catttcacac cctcctgcca
     1381 aagtttagca acgacccgaa ggctctgttt gtcctggacc cgccatacct gtgtacgaaa
     1441 caagagagct acaaacaagc gacctacttt gacctgattg atttcctgcg tctggttaac
     1501 attacgcgtc cgccgtatgt gttcttttcg tccaccaaga gcgagttcat tcgcttcgta
     1561 aactacatgc tggaagataa agtcgataac tggcaggcat ttgagaacgc gaagcgtatt
     1621 accgtgaacg ccaagctgaa ctatcaagtg gcgtatgaag ataatctggt gtataagttc
     1681 ctggagcatc accatcatca ccattgataa ggttagaggt ctcgcggccg ccaccgctga
     1741 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1801 aggaggaact atatccgggt aacgaattca agcttgatat cattcaggac gagcctcaga
     1861 ctccagcgta actggactgc aatcaactca ctggctcacc ttcacgggtg ggcctttctt
     1921 cggtagaagt cttctcatga ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac
     1981 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2041 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2101 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2161 actgttcttc tagtgtagcc gtagttagcc caccacttca agaactctgt agcaccgcct
     2221 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2281 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2341 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2401 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2461 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2521 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2581 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2641 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2701 aaccgtatta ccgcctttga gtgagctgat accgctcgcg cagaaaggcc cacccgaagg
     2761 tgagccaggt gattacattt gggccctcat tagaaaaact catcgagcat caaatgaaac
     2821 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2881 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2941 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3001 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3061 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3121 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg
     3181 ttaaaaggac aattacaaac aggaatcgag tgcaaccggc gcaggaacac tgccagcgca
     3241 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaacgc tgtttttccg
     3301 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3361 ggaagtggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3421 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaag
     3481 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3541 tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat atggctcat
//
